Detailed knowledge of the structural and functional aspects of the molecules involved in the interaction, binding, and fusion of HIV-1 and the closely related SIV with their target cells is fundamental to our understanding of the disease processes in AIDS and to the rational design of diagnostic reagents, vaccines and other therapeutic agents. Two of the most important molecules in the infectious process, namely the envelope glycoproteins (Env) glycoproteins, SU (surface), and TM (transmembrane), are only partially characterized at the structural level with many of their important features still poorly defined. These areas of uncertainty include the orientation of the variable loops, some of the structural changes that occur during the binding and fusion processes, and the features which foster or inhibit Ab-mediated neutralization. Another area of uncertainty involves the distribution Env proteins on viral particles of various types and developmental stages. This proposal describes investigations aimed at applying new approaches for the determination of the 3D morphology of the HIV-1, HIV-2, and SIV envelope (Env) surface (SU, gp120) and transmembrane (TM, gp41) regions as they are expressed on the viral surface and in purified recombinant form. Our goals are to determine the numbers, locations, orientations, and structural details of Env spikes, both alone and in complex with monoclonal antibodies and ligands, on the surface of various forms of HIV and SIV viral particles. In addition we will expand upon our previous epitope and reactive-site mapping studies of individual molecules and complexes by conducting 3D structural analyses on 2D arrays of recombinant soluble trimeric Env proteins and immune complexes. The primary methodologies will be the analysis of intact virions by state-of-the-art cryo-electron microscopic (cryoEM) tomography and computationally assisted model building, including fitting of previoulsy described atomic structures into our 3D models. Relevance to Public Health: The grant proposal is designed to provide key information on the AIDS viral surface proteins that are responsible for infection. These proteins also are the targets of the immune system's antibody defenses against AIDS. The information provided could help in the design of therapeutic agents and vaccines.